Roles of antibodies in AIDS are poorly understood, in part because cell culture definitions of neutralization are inadequate. Although structures of gp120-gp41 complexes with neutralizing antibodies are known, the mechanism(s) by which they lower HIV-1 titers are unproven. Nevertheless, it has been assumed that neutralizing antibodies directly and perhaps irreversibly inactivate HIV-1. We made unique resources (e.g., cell clones with discrete amounts of CD4 and CCR5 including JC.53 cells used to make the TZM-bl derivative) that are widely used for vaccine and entry inhibitor analyses. We also isolated adapted HIV-1 variants that efficiently use damaged CCR5s [e.g., CCR5( 18) lacking the tyrosine-sulfated amino terminus (Nt) and others that efficiently use CCR5( 18) only while the Nt peptide is in the medium]. Recently, we found that infectivity is determined by a race between entry and competing processes that cause viral inactivation. Accordingly, we can alter HIV-1 infectivities by inhibiting the competing processes and/or by changing Nt concentrations and membrane fusion rates. Unexpectedly, we found that the broadly neutralizing 2G12 monoclonal antibody (MAb) reversibly inhibits HIV-1 by slowing entry. Specifically, 2G12 neutralization results indirectly from processes that kinetically compete with infection in the assay conditions, and single virions are increasingly slowed but not irreversibly inactivated when 2G12 concentration increases. To explore these issues, we propose three specific aims. Specific Aim 1 investigates Ab neutralization mechanisms in culture and in vivo: (a) Determine whether widely studied broadly neutralizing MAbs generally slow entry or whether some directly and irreversibly inactivate virions. (b) Analyze the mechanisms of HIV-1 inactivation by Abs relevant to disease control by studying available neutralizing antibodies and escape mutations that coevolved in SHIVSF162P3-infected macaques. (c) Develop new assays to optimally detect these relevant in vivo neutralization mechanisms. Specific Aim 2 dissects roles of CCR5 regions in mediating rates and steps of HIV-1 entry: (a) Isolate adapted HIV-1s that are increasingly reliant on Nt, with the goal of ultimately obtaining variants that can use soluble Nt as coreceptor. (b) Using HIV-1s that efficiently infect HeLa-CD4/CCR5( 18) cells or T-lymphocyte/CCR5( 18) cells only while Nt is present, synchronously induce or block entry of stably adsorbed virions in physiological conditions. Learn which intermediates are sensitive to entry inhibitors and antibodies. Because Nt is membrane impermeable, it provides a unique means to definitively evaluate the role of endocytosis in HIV-1 entry and escape from Abs. Specific Aim 3 determines how envelope glycoprotein redundancy contributes to HIV-1 entry speed and resistance to Ab neutralization. This project uses unique resources and approaches to understand kinetics of HIV-1 infection, mechanisms of viral adaptation to entry limitations including neutralizing Abs, and related issues important for optimizing entry inhibitors and vaccines for AIDS. PUBLIC HEALTH RELEVANCE: This project uses novel methods and unique proven resources to quantitatively investigate HIV-1 invasion of cells and the mechanisms of virus inactivation and escape from antibodies. This work illuminates our understanding of HIV-1 disease and will greatly benefit efforts to develop entry inhibitors and vaccines for AIDS.